Increased reactive oxygen species levels cause ER stress and cytotoxicity in andrographolide treated colon cancer cells

Abstract

Chemotherapy continues to play an essential role in the management of many cancers including colon cancer, the third leading cause of death due to cancer in the United States. Many naturally occurring plant compounds have been demonstrated to possess anti-cancer cell activity and have the potential to supplement existing chemotherapy strategies. The plant metabolite andrographolide induces cell death in cancer cells and apoptosis is dependent upon the induction of endoplasmic reticulum stress (ER stress) leading to the unfolded protein response (UPR). The goal of the present study was to determine the mechanism by which andrographolide induces ER stress and to further evaluate its role in promoting cell death pathways. The T84 and COLO 205 cancer cell lines were used to demonstrate that andrographolide induces increased ROS levels, corresponding anti-oxidant response molecules, and reduced mitochondrial membrane potential. No increases in ROS levels were detected in control colon fibroblast cells. Andrographolide-induced cell death, UPR signaling, and CHOP, Bax, and caspase 3 apoptosis elements were all inhibited in the presence of the ROS scavenger NAC. Additionally, andrographolide-induced suppression of cyclins B1 and D1 were also reversed in the presence of NAC. Finally, Akt phosphorylation and phospho-mTOR levels that are normally suppressed by andrographolide were also expressed at normal levels in the absence of ROS. These data demonstrate that andrographolide induces ER stress leading to apoptosis through the induction of ROS and that elevated ROS also play an important role in down-regulating cell cycle progression and cell survival pathways as well.

Keywords: andrographolide; chemotherapy; endoplasmic reticulum stress; reactive oxygen species; unfolded protein response.

Figure 1. Andrographolide suppresses cell proliferation and cell survival in COLO 205 cells

(A) Cells were treated with Andro for 24, 48 and 72 h and cell viability was quantified by MTT assay. (B) Fluorescence microscopy images showing the viability of COLO 205 cells cultured in vitro with or without Andro (45 μM) (left to right: phase contrast (PC) image, FDA stain, PI stain, overlay of FDA and PI stain). (C) Cells were treated with Andro IC₅₀ dose (45 μM) for either 24 h (upper panels) or 48 h (lower panels) and stained with DAPI. Apoptotic cells were identified by condensation and fragmentation (arrows) of nuclei using inverted fluorescence microscope. (D) Detection of nucleosomes in cytoplasmic fractions at increasing doses of Andro and TM. 10⁴ cells were treated with or without Andro (20, 40, or 60 μM) or TM (1μg/ml) for 48 h at 37°C. Cell lysates (20 μl) were analyzed in the ELISA. (E) Caspase 3 activity was evaluated by Caspase-3 colorimetric activity assay kit as described. Experiments were performed two times (D and E) or three times (A, B and C). (*P < 0.05, ***P < 0.001).

Figure 2. Andrographolide induces ROS generation in colon cancer cells

(A) The effects of Andro treatment on ROS generation. T84, COLO 205, and normal fibroblast cells (18Co) were treated with Andro IC₅₀ (45 μM) for 24 h and cellular ROS levels were determined by measurement of fluorescent DCF. ROS inhibition was performed by pre-treatment of cells with 20 mM NAC for 1 h prior to Andro treatment. (B) The effects of ROS on cell viability in Andro-treated colon cancer cells were assessed by MTT assay at 24 h and 48 h in the presence of absence of NAC. Absorbance was read at 570 nm with averages from triplicate wells. (C) For quantification of clonogenicity Andro treated T84 and COLO 205 cells were examined by colony formation assay for 24 h and 48 h and visualized by staining with crystal violet. (D) T84 and COLO 205 cells were treated with or without Andro for 24 h and 48 h and then incubated with TMRE. Experiments were performed two times (A and D) or three times (B and C). (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 3. Andrographolide induces anti-oxidant gene expression

T84 and COLO 205 cells were treated with or without Andro at IC₅₀ (45 μM) for 4, 6, or 8 h and the transcriptional level of expression for anti-oxidant genes (LPO, Nrf-2, Trx, GPx, Prx-6) were determined by qRT-PCR for (A) T84 cells and (B) COLO 205 cells. Bar graphs show quantitative results normalized to GAPDH mRNA levels. Results are from three independent experiments. (C) T84 and COLO 205 cells were grown on coverslips and treated with Andro. TRX expression was evaluated by immunofluorescent staining. Nuclei were stained using DAPI and cells were examined by fluorescence microscopy. Fluoresence intensity was determined and compared with untreated (Con) T84 and COLO 205 cells. (D) T84 and COLO 205 cells were treated with Andro for 4 h, 6 h and 8 h. Cells were lysed and protein expression was determined by immunoblotting for PrX, GPX and GAPDH. Densitometry analysis was performed and normalized with GAPDH. Statistical significance was determined using one way-ANOVA followed by post hoc Tukey's test. (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 4. Andrographolide mediated ER stress and apoptosis signaling are dependent on increased ROS

(A) T84 and COLO 205 cells were pre-treated with or without NAC followed by Andro IC₅₀ (45 μM) for 24 h or 48 h. Cells were lysed and protein expression for IRE-1 and GAPDH were evaluated by immunoblot. Densitometry analysis was performed and normalized with GAPDH. Cells were also evaluated for mRNA expression by qRT-PCR for (B) IRE-1 and pro-apoptosis downstream signaling proteins, (C) CHOP, and (D) XBP-1. Results shown are from three independent experiments (*P < 0.05, **P < 0.01, ***P < 0.001).

Figure 5. Andrographolide induced cell cycle arrest and apoptosis in colon cancer cells is ROS dependent

(A) T84 and COLO 205 cells were pretreated with or without NAC followed by Andro IC₅₀ (45 μM) for 24 h and 48 h. Cell lysates were analyzed by immunoblot and quantified by densitometry for expression of Cyclin B1 and cyclin A. Expression is normalized against GAPDH expression. (B) T84 and COLO 205 cells were pretreated with or without NAC followed by Andro IC₅₀ for 24 h and 48 h. Cells were then lysed and incubated with caspase 3 substrate and activity was determined by colorimetric assay at OD_405nm. (**P < 0.01, ***P < 0.001).

Figure 6. Andrographolide induced oxidative stress inhibits the Akt/mTOR cell survival signaling pathway

(A–B) Cell lysates from T84 and COLO 205 cells pretreated with or without NAC and then treated with Andro IC₅₀ (45 μM) for 48 h were analyzed by immunoblot for expression of phospho-Akt (ser473 and Thr 308), total Akt and GAPDH and quantified by densitometry. (C) T84 and COLO 205 cells were grown on coverslips and pretreated with or without NAC, followed by Andro IC₅₀ for 48 h and then phospho-mTOR expression was evaluated by immunofluorescent staining. Nuclei were stained using DAPI and cells were examined by fluorescence microscopy. Fluorescence intensity was determined and compared with untreated T84 and COLO 205 cells.